... cells were washed with NaCl ⁄ Piand fixed with 2% formaldehyde and 0.2% glutaradehyde at room temper-ature for 15 min. The cells were then washed with NaCl ⁄ Piand permeabilized with 2 mm MgCl2, ... phalloidin (Invitrogen) was added for F-actin staining at room temperature for 45 min. Finally, thecells were washed three times with 1· NaCl ⁄ Pibefore micros-copy was performed using the 20· objective ... RT-PCRanalysis was performed as described previously. Theexpected sizes of the PCR products were 1270 bp for HVEM, 738 bp for nectin-1, 616 bp for nectin-2, and736 bp for 3-OST-3, respectively....